Observation method and holder for gel-like transparent sample which encloses an observation target

ABSTRACT

Provided is a sample observation method including: bringing a gel-like transparent sample that encloses an observation target into contact with a transparent flat surface section of a substrate; and collecting light from the observation target by means of an objective lens via the substrate, in a state in which a pressing force is made to act in a direction in which the sample and the flat surface section relatively approach each other, until the contact area between the sample and the flat surface section comes to have a size allowing an effective light flux for the objective lens of a microscope to pass therethrough.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on Japanese Patent Application No.2017-218710, the content of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a sample observation method and asample holder.

BACKGROUND ART

In recent years, a method has been focused, in which microscopic imagedata of a 3D-cultured cell, such as cellular aggregates, is obtained,screening is performed by using an image analysis technology, and drugefficacy is evaluated. Known as a method for forming a cellularaggregate, for example, is a method in which cells are dispensed,together with a culture solution, to an inner surface of a lid of apetri dish in the form of a droplet, this droplet is inverted to form ahanging drop, and cell aggregation is caused inside the hanging dropwith the help of a component force of gravity, in the direction alongthe curved surface of the hanging drop (for example, see PTL 1).However, because hanging drops are not subjected to accurate arrayarrangement, it is clear that this method is not suitable for automationof preparation of cellular aggregates.

There is a known multiwall-plate structure that allows hanging dropssuitable for automation to be formed, by improving this issue of PTL 1(for example, see PTL 2). The multiwell plate described in PTL 2 isformed by arranging, in an array, sets each of which includes a hollowsection that receives a liquid discharged from a dispenser, ahanging-drop forming compartment that forms and holds a hanging drop,and a duct that leads to the hollow section and the hanging-drop formingcompartment. In the multiwell plate described in PTL 2, it is notnecessary to invert droplets, unlike the method described in PTL 1, andhanging drops can be formed merely by dispensing cells and a culturesolution from above the multiwell plate according to an arrayarrangement format, thereby facilitating automation of preparation ofcellular aggregates in hanging drops. However, PTL 2 does not mentionnothing about a high-definition observation method for a microscope, asin PTL 1.

Furthermore, because the shape of a hanging drop is formed of a curvedsurface, if the hanging drop is observed by means of a microscope, thecurved surface shape of the hanging drop acts as a lens due to therefractive-index difference between the hanging drop and a substance(for example, air when a dry objective is used) that is interposedbetween the hanging drop and an objective lens, and optical aberrationsoccur, thus making it impossible to acquire a high-definitionobservation image. PTLs 1 and 2 do not mention nothing about a solutionto this issue.

An adverse effect of the refractive-index difference on the observationprominently appears particularly when the substance interposed betweenthe hanging drop and the objective lens is air. Thus, it is conceivablethat the hanging drop is gelled, and the gelled hanging drop is immersedin a liquid and is observed, thereby reducing the refractive-indexdifference and suppressing the occurrence of aberrations; however,because the refractive indices of the liquid and the hanging drop gelare also strictly different, the original optical performance of themicroscope cannot be obtained.

There is a known technique for achieving fine observation performed by amicroscope and automation of image acquisition and the observation, byfurther developing the technique of PTL 2 (for example, see PTL 3). Withthe technique described in PTL 3, prepared cellular aggregates inhanging drops are dropped, together with the hanging drops, on wells ofa multiwell plate, the bottom surfaces of the wells being flat, andlight produced by each of the cellular aggregates is collected by anobjective lens of an inverted microscope via the bottom surface of thecorresponding well, thereby performing observation and imageacquisition. The wells are designed with ingenuity; specifically, thetransverse section of each of the wells is gradually narrowed downward,and the bottom surface of the well has a slightly larger size than thecellular aggregate (for example, 100 to 500 μm), for example, a shapehaving a size of about 1 mm in diameter, thereby making it possible toalmost fix the XY-positions of the cellular aggregate dropped on thebottom surface (the positions in the directions intersecting thevertical direction). Accordingly, automation of the observation isachieved, and the influence of aberrations occurring on the curvedsurface of the hanging drop is reduced.

CITATION LIST Patent Literature

{PTL 1} DE patent invention No. 10362002 specification

{PTL 2} Publication of Japanese Patent No. 5490803

{PTL 3} PCT International Publication No. WO 2017/001680

SUMMARY OF INVENTION

A first aspect of the present invention is directed to a sampleobservation method including: bringing a gel-like transparent samplethat encloses an observation target into contact with a transparent flatsurface section of a substrate; and collecting light from theobservation target by means of an objective lens via the substrate, in astate in which a pressing force is made to act in a direction in whichthe sample and the flat surface section relatively approach each other,until the contact area between the sample and the flat surface sectioncomes to have a size allowing an effective light flux for the objectivelens of a microscope to pass therethrough.

A second aspect of the present invention is directed to a sampleobservation method including: bringing a gel-like transparent samplethat encloses an observation target into contact with an objective lensof a microscope; and collecting light from the observation target bymeans of the objective lens, in a state in which a pressing force ismade to act in the direction in which the sample and the objective lensrelatively approach each other, until the contact area between thesample and the objective lens comes to have a size allowing an effectivelight flux for the objective lens to pass therethrough.

A third aspect of the present invention is directed to a sample holderincluding: a substrate that has a transparent flat surface section withwhich a gel-like transparent sample that encloses at least oneobservation target is brought into contact; and a contact-statemaintaining unit that maintains a contact state in which the contactarea between the sample and the flat surface section has a size allowingan effective light flux for an objective lens of a microscope to passtherethrough.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a flowchart for explaining a sample observation methodaccording to a first embodiment of the present invention.

FIG. 2 is a longitudinal sectional view of a sample holder according tothe first embodiment of the present invention.

FIG. 3 is a longitudinal sectional view of a sample holder using amultiwell plate, according to a modification of the first embodiment ofthe present invention.

FIG. 4 is a longitudinal sectional view of a sample holder using amultiwell plate, according to a first modification of the firstembodiment of the present invention.

FIG. 5A is a longitudinal sectional view showing an example deep wellfor culturing or transparentizing, used in a sample observation methodaccording to a second modification of the first embodiment of thepresent invention.

FIG. 5B is a longitudinal sectional view showing an example shallow wellfor observation, used in the sample observation method according to thesecond modification of the first embodiment of the present invention.

FIG. 6 is a longitudinal sectional view of a sample holder using a slideglass, according to a third modification of the first embodiment of thepresent invention.

FIG. 7 is a longitudinal sectional view of a hanging-drop forming deviceetc. for dropping a hanging drop by a sample observation methodaccording to a second embodiment of the present invention.

FIG. 8 is a longitudinal sectional view of a sample holder according toa modification of the second embodiment of the present invention.

FIG. 9 is a plan view for explaining a state in which a cellularaggregate in a droplet on a rod is observed, by a sample observationmethod according to a third embodiment of the present invention.

DESCRIPTION OF EMBODIMENTS First Embodiment

A sample observation method and a sample holder according to a firstembodiment of the present invention will be described below withreference to the drawings.

As shown in the flowchart of FIG. 1 and in FIG. 2, the sampleobservation method of this embodiment includes: a step S1 of bringing agel-like transparent hanging drop (sample) D that encloses at least onecellular aggregate (observation target) S into contact with atransparent flat bottom section (flat surface section) 3 a of a well(substrate) 3; and a step S2 of collecting, in this state, light fromthe cellular aggregate S by means of an objective lens 9, detecting thelight, and observing the cellular aggregate S.

The hanging drop D is, for example, in a state in which a droplet of atransparent sodium alginate solution A encloses at least one cellularaggregate S and is hung, at the time of gelation. Furthermore, forexample, when a calcium solution is sprayed on the surface of thedroplet of the sodium alginate solution A, thus being brought intocontact therewith, the hanging drop D becomes a gel up to the vicinityof the periphery of the enclosed cellular aggregate S through a chemicalreaction. The specific gravity of the sodium alginate solution A is 1and is less than the specific gravity of the cellular aggregate S.

As shown in FIG. 2, for example, this sample observation method isperformed by using a sample holder 7 that is provided with: ahanging-drop forming device (base material) 1 that forms and supportsthe hanging drop D; the well 3, which can accommodates the hanging dropD; and a positioning mechanism (contact-state maintaining means) 5 thatmaintains the hanging-drop forming device 1 in a state in which thehanging drop D and the bottom section 3 a of the well 3 are brought intocontact with each other.

The hanging-drop forming device 1 is provided with: a hollow section 11into which the solution is injected; a hanging-drop forming compartment13 that supports a droplet of the solution injected into the hollowsection 11, in a hung state, with a cellular aggregate S being enclosedtherein; and a thin duct 15 that connects the hollow section 11 and thehanging-drop forming compartment 13.

The hanging-drop forming device 1 may be constituted of one set of thehollow section 11, the hanging-drop forming compartment 13, and the duct15 or may be a multiwell plate that is formed by arranging such sets inan array. FIG. 2 shows a hanging-drop forming device 1 that is formed ofone set of the hollow section 11, the hanging-drop forming compartment13, and the duct 15.

The hollow section 11 has an opening 11 a that opens toward the oppositeside from the duct 15 and has a substantially conical shape that isnarrowed in a tapered manner from the opening 11 a and that leads to theduct 15.

The hanging-drop forming compartment 13 has a substantially conicalshape that gradually expands radially outward from the duct 15. Thehanging-drop forming compartment 13 supports the droplet of the sodiumalginate solution A, with a lower section thereof being exposed.

The duct 15 has a through-hole 15 a that penetrates from the hollowsection 11 to the hanging-drop forming compartment 13.

Furthermore, the hanging-drop forming device 1 is further provided witha flange 17 that bulges radially outward from the hollow section 11. Theflange 17 supports the hollow section 11 and the hanging-drop formingcompartment 13, with the hollow section 11 facing vertically upward andthe hanging-drop forming compartment 13 facing vertically downward.Furthermore, the flange 17 has a fitting section 17 a into which anopening 3 b of the well 3 can be fitted, and can be disposed onto theopening 3 b of the well 3 in a fitted state. Hereinafter, the verticaldirection is referred to as Z-direction, and directions that intersectthe Z-direction and that are perpendicular to each other are referred toas X-direction and Y-direction.

The well 3 is formed of a transparent material through which light canbe transmitted. A transparentizing solution (liquid) W1 for making thehanging drop D and the cellular aggregate S transparent is accumulatedin the well 3, for example. Furthermore, the well 3 is supported by astage 19.

The positioning mechanism 5 is provided with an elastic member 21, suchas rubber, that is attached to the opening 3 b of the well 3 and thatpositions the flange 17 of the hanging-drop forming device 1, into whichthe opening 3 b is fitted. The section of the opening 3 b of the well 3where the elastic member 21 is disposed is formed to be thicker than theother section of the opening 3 b. In the positioning mechanism 5, whenthe elastic member 21 is fitted by means of the flange 17, the elasticmember 21 is brought into close contact with an inner surface of thefitting section 17 a of the flange 17 due to the elastic force thereof,thus making it possible to position the flange 17.

In Step S1, the hanging drop D, which is supported by the hanging-dropforming device 1, is immersed in the transparentizing solution W1accumulated in the well 3. Then, the hanging drop D is brought intocontact with the bottom section 3 a of the well 3, a pressing force ismade to act between the hanging drop D and the bottom section 3 a of thewell 3 in a direction in which they relatively approach each other,until the contact area between the hanging drop D and the bottom section3 a of the well 3 comes to have a size allowing an effective light fluxfor the objective lens 9 to pass therethrough, and the positioningmechanism 5 maintains that state.

In Step S2, for example, by means of an inverted light sheet microscope(not shown), excitation light that is collected in a planar manner alonga plane perpendicular to the vertical direction is made incident from alateral side of the hanging drop D and is radiated onto the cellularaggregate S. Then, of fluorescence that is produced in the cellularaggregate S, fluorescence that is emitted vertically downward from thelower section of the hanging drop D is collected by the objective lens 9and is detected.

An immersion objective lens is used as the objective lens 9, forexample. An immersion solution W2 is held due to a surface tension in agap between a distal end of the objective lens 9 and the bottom section3 a of the well 3. By adopting the immersion objective lens, ahigher-resolution observation image can be acquired.

The operation of the thus-configured sample observation method andsample holder 7 will now be described.

In order to observe a hanging drop D by using the sample observationmethod and the sample holder 7 of this embodiment, first, a gel-likehanging drop D that encloses a cellular aggregate S is formed by meansof the hanging-drop forming device 1.

Specifically, cells (not shown) are dispensed, together with the sodiumalginate solution A, to the hollow section 11 of the hanging-dropforming device 1 and are gravitationally moved to the hanging-dropforming compartment 13 via the through-hole 15 a of the duct 15, thusbeing formed into a hanging drop D that is in a hung state of a dropletof the sodium alginate solution A enclosing the cells. Then, the cellsare cultured inside the hanging drop D and are formed into a cellularaggregate S that is an observation target.

Because the sodium alginate solution A, which forms the hanging drop D,is less in specific gravity than the cellular aggregate S, the cellularaggregate S gravitationally settles down in the vicinity of the lowestpoint of the hanging drop D. Therefore, the amount of the sodiumalginate solution A to be dispensed to the hollow section 11 isdetermined in advance, thereby making it possible to fix not only thepositions of the cellular aggregate S in the X-direction and theY-direction but also the position thereof in the Z-direction.Furthermore, by using the hanging-drop forming device 1, it is notnecessary to invert the droplet of the sodium alginate solution A, inorder to form the hanging drop D.

Next, a calcium solution is nebulized by a nebulizer device (not shown)or the like that nebulizes the calcium solution with ultrasound orthrough pressurization, for example, the nebulized calcium solution isbrought into contact with the surface of the hanging drop D, which issupported by the hanging-drop forming device 1, and is made to penetratethe inside thereof, and the sodium alginate solution A is made to becomea gel up to the vicinity of the periphery of the enclosed cellularaggregate S. Accordingly, a sample in which the position of the cellularaggregate S is fixed in the substantially transparent hanging drop D isprepared.

Then, as shown in FIG. 1, the hanging drop D, which is supported by thehanging-drop forming device 1, is immersed in the transparentizingsolution W1 accumulated in the well 3, and transparentizing processingfor making the hanging drop D and the cellular aggregate S transparentis applied thereto. The hanging drop D is immersed in a solution, suchas the transparentizing solution W1, thereby making it also possible tosuppress drying of the hanging drop D during observation.

Note that, when a large cellular aggregate S whose diameter exceeds 300μm is observed, excitation light for observation does not reach theinside of the cellular aggregate S, thus making it impossible to observethe internal structure of the cellular aggregate S, in some cases. Byapplying the transparentizing processing, excitation light forobservation can be made to easily reach the inside of the cellularaggregate S even when the cellular aggregate S is large. Accordingly,the internal structure of the cellular aggregate S can be easilyobserved, irrespective of the size of the cellular aggregate S.

Next, the lower section of the hanging drop D is brought into contactwith the bottom section 3 a of the well 3, and the hanging-drop formingdevice 1 is pushed to cause the elastic member 21 on the well 3 to befitted by means of the flange 17 such that the contact area therebetweencomes to have a size allowing an effective light flux for the objectivelens 9 of the microscope to pass therethrough. Accordingly, the flange17 is positioned due to the elastic force of the elastic member 21, thusmaintaining a state in which the contact area between the hanging drop Dand the bottom section 3 a of the well 3 has a size allowing aneffective light flux for the objective lens 9 of the microscope to passtherethrough.

Next, in this state, planar excitation light is made to enter the well 3from the outside and is radiated onto the cellular aggregate S in thehanging drop D from a lateral side of the hanging drop D by means of thelight sheet microscope, and fluorescence that is emitted verticallydownward from the cellular aggregate S is collected by the objectivelens 9 and is detected.

In this case, because the hanging drop D and the bottom section 3 a ofthe well 3 are brought into contact with each other, and the contactarea therebetween is maintained to have a size allowing an effectivelight flux for the objective lens 9 to pass therethrough, fluorescencethat is produced in the cellular aggregate S in the hanging drop D isdirectly transmitted through the bottom section 3 a of the well 3,without being transmitted through external air or liquid, and iscollected by the objective lens 9.

Accordingly, it is possible reduce the refractive-index difference inthe light path from the hanging drop D to the objective lens 9, toprevent the hanging drop D from acting as a lens even when the hangingdrop D has a curved surface shape, and to suppress the occurrence ofoptical aberrations. For example, even in a case in which the gel-likesodium alginate solution A has a refractive index of about 1.33, and thetransparentizing solution W1 has a refractive index of about 1.45, theoccurrence of an optical deterioration can be prevented without beingaffected by the refractive-index difference therebetween. Furthermore,because it is a simple task to merely maintain a state in which thegel-like hanging drop D enclosing the cellular aggregate S and thebottom section 3 a of the well 3 are brought into contact with eachother, observation of the cellular aggregate S can be automated.

Therefore, according to the sample observation method and the sampleholder 7 of this embodiment, it is possible to get the best from theoriginal optical performance of the microscope, to enablehigh-definition observation and image acquisition of the cellularaggregate S performed by the microscope, and to facilitate automation ofthe observation.

Note that observation is performed by means of the light sheetmicroscope, thereby matching the focal position of the excitation lightin the cellular aggregate S with a detection optical axis, and the focalplane of the objective lens 9 is matched with the incident plane of theexcitation light, thereby making it possible to collect fluorescencethat is produced in a wide range extending along the focal plane of theobjective lens 9 at once by means of the objective lens 9, and to easilyacquire a clear fluorescence image of an observation site of thecellular aggregate S.

In this embodiment, as shown in FIG. 3, as the hanging-drop formingdevice 1, it is also possible to adopt a multiwell plate on which setsof the hollow sections 11, the hanging-drop forming compartments 13, andthe ducts 15 are arranged in an array. By doing so, automatic dispensingis easy, and image acquisition of a large number of cellular aggregatesS for the purpose of screening can be performed by high-throughput.

This embodiment can be modified as follows.

In this embodiment, although the elastic member 21 is adopted as thepositioning mechanism, in a first modification, for example, as shown inFIG. 4, the hanging drop D may be pressed against the bottom section 3 aof the well 3 due to the self-weights of the hanging-drop forming device1 and the flange 17, so as to ensure a contact area having a sizeallowing an effective light flux for the objective lens 9 to passtherethrough. FIG. 4 shows an example case in which a multiwell plate onwhich sets of the hollow sections 11, the hanging-drop formingcompartments 13, and the ducts 15 are arranged in an array of 3×4 isadopted.

Furthermore, in a second modification, as shown in FIGS. 5A and 5B, twotypes of wells 3′ and 3″ that have different depths are prepared assubstrates. The deep well 3′, in which the hanging drop D is not broughtinto contact with the bottom section 3 a, such as that shown in FIG. 5A,may be used when cells are cultured in the hanging drop D or when thehanging drop D and the cellular aggregate S are made to be transparent,and the shallow well 3″, in which a sufficient contact area between thehanging drop D and the bottom section 3 a can be ensured, such as thatshown in FIG. 5B, may be used at the time of observation.

In this case, for example, it is also possible to provide a projectionor a depression on the inner surface of the fitting section 17 a of theflange 17, to provide a depression or a projection on the outer surfaceof the opening 3 b of the shallow well 3″, which is used forobservation, shown in FIG. 5B, and to make these projection anddepression function as a notch mechanism (positioning mechanism) 23.Then, the notch mechanism 23 enables to maintain a state in which thehanging drop D is pushed to ensure a sufficient contact area between thehanging drop D and the bottom section 3 a of the well 3″.

Furthermore, in a third modification, the hanging drop D may not beimmersed in the transparentizing solution W1 in the well 3.

In this case, for example, as shown in FIG. 6, it is also possible toadopt, as the substrate, a slide glass 25 that has a transparent flatsurface section 25 a, to press the hanging drop D, which is supported bythe hanging-drop forming device 1, against the flat surface section 25 afrom above the slide glass 25, and to ensure a contact area having asize allowing an effective light flux for the objective lens 9 to passtherethrough. In this case, for example, it is also possible to supportthe slide glass 25 by means of the stage 19, to grasp the flange 17 bymeans of a robot hand (positioning mechanism) 27 or the like, and tomaintain a contact state between the hanging drop D and the flat surfacesection 25 a of the slide glass 25.

Second Embodiment

Next, a sample observation method and a sample holder according to asecond embodiment of the present invention will be described.

The sample observation method and the sample holder of this embodimentdiffer from those of the first embodiment in that a slide glass 25 thathas a transparent flat surface section 25 a is adopted as the substrate,instead of the well 3, and the hanging drop D is dropped on the slideglass 25, thus bringing the hanging drop D into contact with the flatsurface section 25 a, as shown in FIG. 7, for example.

Hereinafter, identical reference signs are assigned to configurationscommon to those in the sample observation method and the sample holder 7of the first embodiment, and a description thereof will be omitted.

As shown in FIG. 7, in the sample observation method of this embodiment,the hanging-drop forming device 1, which supports the hanging drop D, isvibrated by a vibrator 29, such as an ultrasound transducer, thusdropping the hanging drop D on the slide glass 25. Accordingly, agel-like transparent droplet (sample) B of the sodium alginate solutionA, which had formed the hanging drop D, and the flat surface section 25a of the slide glass 25 are brought into contact with each other, andthe contact area between the droplet B and the flat surface section 25 ais formed due to the self-weight of the droplet B.

Then, in a state in which the contact area with the bottom section 3 aof the well 3 is maintained to have a size allowing an effective lightflux for the objective lens 9 to pass therethrough, due to theself-weight of the droplet B of the sodium alginate solution A, lightfrom the cellular aggregate S enclosed in the droplet B is collected bythe objective lens 9 and is detected, and the cellular aggregate S isobserved.

Therefore, according to the sample observation method of thisembodiment, in a case in which the flexibility of the gel-like hangingdrop D is high, a contact area having a sufficient size allowing aneffective light flux for the objective lens 9 to pass therethrough canbe formed between the dropped gel-like droplet B and the flat surfacesection 25 a of the slide glass 25, by a simple method in which thehanging drop D is merely dropped on the slide glass 25.

In this embodiment, as shown in FIG. 8, it is also possible to adopt, asthe contact-state maintaining means, a pressing member (contact-statemaintaining means) 31 that presses the gel-like droplet B of the sodiumalginate solution A against the flat surface section 25 a of the slideglass 25 in the pressing direction. The slide glass 25 and the pressingmember 31 constitute a sample holder 33.

By doing so, even in a case in which a sufficient contact area with theflat surface section 25 a of the slide glass 25 cannot be ensured onlydue to the self-weight of the gel-like droplet B, it is possible topress the gel-like droplet B against the flat surface section 25 a ofthe slide glass 25 by means of the pressing member 31, to ensure acontact area having a size allowing an effective light flux for theobjective lens 9 to pass therethrough, and to maintain that state.

Third Embodiment

Next, a sample observation method according to a third embodiment of thepresent invention will be described.

The sample observation method and the sample holder of this embodimentdiffer from those of the first embodiment in that the gel-liketransparent droplet (sample) B of the sodium alginate solution A isbrought into contact with the objective lens 9, without adopting thesubstrate, as shown in FIG. 9, for example.

Hereinafter, identical reference signs are assigned to configurationscommon to those in the sample observation methods of the firstembodiment and the second embodiment, and a description thereof will beomitted.

In the sample observation method of this embodiment, as shown in FIG. 9,a dispenser 35 is used to dispense the sodium alginate solution A,together with cells, on an end face 37 a of a rod 37, thus forming adroplet B on the end face 37 a, and the cells are cultured in thedroplet B to form a cellular aggregate S that is an observation target.Then, a calcium solution is sprayed on the droplet B on the end face 37a of the rod 37, to make the calcium solution penetrate inside thereof,thus making the droplet B become a gel up to the vicinity of theperiphery of the enclosed cellular aggregate S.

Next, an objective lens 9 that has an optical design conforming to therefractive index of the gel-like sodium alginate solution A is disposedso as to be directed vertically downward, and the rod 37 is moved byusing a movement mechanism (not shown) to press the gel-like droplet Bagainst the objective lens 9 from below. Then, in a state in which thepressing force is made to act in the direction in which the gel-likedroplet B approaches the objective lens 9, until the contact areabetween the gel-like droplet B and the objective lens 9 comes to have asize allowing an effective light flux for the objective lens 9 to passtherethrough, fluorescence from the cellular aggregate S is collected bythe objective lens 9, and the cellular aggregate S is observed.

In this case, due to the elastic force of the gel-like droplet B, thepressing amount is changed within the range of the elastic force,thereby making it possible to change the focal point of the objectivelens 9 in the optical-axis direction. Then, observation is performedwhile maintaining a state in which the contact area between the gel-likedroplet B and the objective lens 9 has a size allowing an effectivelight flux for the objective lens 9 to pass therethrough, thereby makingit possible to reduce the refractive-index difference in the light pathfrom the gel-like droplet B to the objective lens 9, to prevent thedroplet B from acting as a lens even when the gel-like droplet B has acurved surface shape, and to suppress the occurrence of opticalaberrations.

Therefore, according to the sample observation method of thisembodiment, it is possible to get the best from the original opticalperformance of the microscope, without using the substrate, such as thewell 3, to enable high-definition observation and image acquisition ofthe cellular aggregate S performed by the microscope, and to facilitateautomation of the observation.

Although the embodiments of the present invention have been described indetail above with reference to the drawings, the specific configurationsare not limited to the embodiments, and design changes etc. that do notdepart from the scope of the present invention are also encompassed. Forexample, without being limited to those applied to the above-describedembodiments and modifications, the present invention can also be appliedan embodiment obtained by appropriately combining these embodiments andmodifications and is not particularly limited.

Furthermore, for example, in the first embodiment, the well 3 has thebottom section 3 a, which is transparent over the whole area thereof,and, in the second embodiment, the slide glass 25 has the flat surfacesection 25 a, which is transparent over the whole area thereof; however,the bottom section of the well 3 and the flat surface section of theslide glass 25 do not need to be transparent over the whole areasthereof, and the bottom section of the well 3 and the slide glass 25 mayeach have partially a transparent flat surface section having a sizeallowing fluorescence from the cellular aggregate S to be transmitted.

The following aspects are also derived from the above-describedembodiments.

According to a first aspect, the present invention provides a sampleobservation method including: bringing a gel-like transparent samplethat encloses an observation target into contact with a transparent flatsurface section of a substrate; and collecting light from theobservation target by means of an objective lens via the substrate, in astate in which a pressing force is made to act in a direction in whichthe sample and the flat surface section relatively approach each other,until the contact area between the sample and the flat surface sectioncomes to have a size allowing an effective light flux for the objectivelens of a microscope to pass therethrough.

According to this aspect, the sample and the flat surface section of thesubstrate are brought into contact with each other, and a state in whichthe contact area therebetween has a size allowing an effective lightflux for the objective lens of the microscope to pass therethrough ismaintained, thus causing light produced in the observation target in thesample to be directly transmitted through the flat surface section ofthe substrate from the sample, without being transmitted throughexternal air or liquid, and to be collected by the objective lens.

Accordingly, it is possible reduce the refractive-index difference inthe light path from the sample to the objective lens, to prevent thesample from acting as a lens even when the sample has a curved surfaceshape, and to suppress the occurrence of optical aberrations.Furthermore, because it is a simple task to merely maintain a state inwhich the gel-like sample enclosing the observation target and the flatsurface section of the substrate are brought into contact with eachother, observation of the sample can be automated. Therefore, it ispossible to get the best from the original optical performance of themicroscope, to enable high-definition observation and image acquisitionof the observation target performed by the microscope, and to facilitateautomation of the observation.

According to a second aspect, the present invention provides a sampleobservation method including: bringing a gel-like transparent samplethat encloses an observation target into contact with an objective lensof a microscope; and collecting light from the observation target bymeans of the objective lens, in a state in which a pressing force ismade to act in the direction in which the sample and the objective lensrelatively approach each other, until the contact area between thesample and the objective lens comes to have a size allowing an effectivelight flux for the objective lens to pass therethrough.

According to this aspect, the sample and the objective lens are broughtinto contact with each other, and a state in which the contact areatherebetween has a size allowing an effective light flux for theobjective lens to pass therethrough is maintained, thus causing lightproduced in the observation target in the sample to be directly incidenton the objective lens from the sample, without being transmitted throughexternal air or liquid, and to be collected.

Accordingly, it is possible to reduce the refractive-index difference inthe light path from the sample to the objective lens, to prevent thesample from acting as a lens even when the sample has a curved surfaceshape, and to suppress the occurrence of optical aberrations.Furthermore, because it is a simple task to merely maintain a state inwhich the gel-like sample enclosing the observation target and theobjective lens are brought into contact with each other, observation ofthe sample can be automated. Therefore, it is possible to get the bestfrom the original optical performance of the microscope, to enablehigh-definition observation and image acquisition of the observationtarget performed by the microscope, and to facilitate automation of theobservation.

In the above-described first aspect, a base material may support thesample in a state in which the sample is brought into contact with theflat surface section.

With this configuration, the base material can maintain a state in whichthe sample is brought into contact with the substrate, in a stableorientation.

In the above-described first aspect, the sample may be dropped on thesubstrate, thus being brought into contact with the flat surfacesection.

With this configuration, the contact area between the sample and thesubstrate can be formed due to the self-weight of the sample.

In this case, the base material may be vibrated from the state in whichthe base material supports the sample, thereby dropping the sample.

With this configuration, the sample, which is supported by the basematerial, can be dropped on the substrate by a simple method.

In the above-described first aspect, the substrate may have a formcapable of accumulating a liquid therein; and the sample and the flatsurface section may be brought into contact with each other in a statein which at least one section of the sample is immersed in the liquidaccumulated in the substrate.

With this configuration, it is possible to suppress drying of the sampleduring observation.

In this case, the liquid is a transparentizing solution for making thesample transparent.

With this configuration, it is possible to facilitate observation of theobservation target enclosed in the sample.

In each of the above-described aspects, the pressing force may be madeto occur due to gravity.

With this configuration, a member for forming the contact area betweenthe sample and the substrate or the objective lens becomes unnecessary,thus making it possible to simplify the configuration. Thisconfiguration is effective when the gel-like sample has a sufficientflexibility.

In each of the above-described aspects, the microscope may be aninverted microscope.

In the above-described aspect, the objective lens may be an immersionobjective lens.

By adopting an immersion objective lens, it is possible to acquire ahigher-resolution observation image.

In each of the above-described aspects, the sample may be formed bycausing a gel-like transparent droplet to gel in a state in which theobservation target is enclosed therein.

In each of the above-described aspects, the sample is formed by causinga hanging drop to gel, the hanging drop being in a state in which thedroplet is hung.

With this configuration, a sample in which the position of theobservation target is fixed in the transparent hanging drop is prepared.Therefore, light produced in the observation target in the sample isdetected outside the sample, thus making it possible to observe theobservation target with high-definition.

In each of the above-described aspects, the observation target is acellular aggregate formed when a plurality of cells aggregate.

According to a third aspect, the present invention provides a sampleholder including: a substrate that has a transparent flat surfacesection with which a gel-like transparent sample that encloses at leastone observation target is brought into contact; and a contact-statemaintaining unit that maintains a contact state in which the contactarea between the sample and the flat surface section has a size allowingan effective light flux for an objective lens of a microscope to passtherethrough.

According to this aspect, the sample and the flat surface section of thesubstrate are brought into contact with each other, and thecontact-state maintaining means maintains a contact state in which thecontact area therebetween has a size allowing an effective light fluxfor the objective lens of the microscope to pass therethrough, therebymaking it possible to cause light produced in the observation target inthe sample to be directly transmitted through the flat surface sectionof the substrate from the sample, without being transmitted throughexternal air or liquid, and to be collected by the objective lens.

Accordingly, it is possible to reduce the refractive-index difference inthe light path from the sample to the objective lens, to prevent thesample from acting as a lens even when the sample has a curved surfaceshape, and to suppress the occurrence of optical aberrations.Furthermore, because it is a simple task to merely maintain a state inwhich the gel-like sample enclosing the observation target and thesubstrate are brought into contact with each other, observation of thesample can be automated. Therefore, it is possible to get the best fromthe original optical performance of the microscope, to enablehigh-definition observation and image acquisition of the observationtarget performed by the microscope, and to facilitate automation of theobservation.

In the above-described third aspect, the contact-state maintaining unitmay be a pressing member that presses the sample in a direction in whichthe sample is pressed against the flat surface section.

With this configuration, even in a case in which a sufficient contactarea between the sample and the substrate cannot be ensured merely bythe self-weight of the sample, a sufficient contact area can be ensuredby pressing the sample against the flat surface section of the substrateby means of the pressing member.

The above-described third aspect may further include a base materialthat supports the sample in a state in which the sample is brought intocontact with the flat surface section.

With this configuration, it is possible to form the contact area betweenthe sample and the substrate, with the sample being in a stableorientation.

In the above-described third aspect, the base material can support adroplet in a hung state, with the droplet enclosing the observationtarget; and the sample is formed by causing the droplet, which issupported by the base material, to gel.

With this configuration, a sample that is in a hung state and in whichthe position of the enclosed observation target is fixed is prepared.Therefore, light produced in the observation target in the sample isdetected outside the sample, thus making it possible to observe theobservation target with high-definition.

In the above-described third aspect, the substrate may have a formcapable of accumulating a liquid therein and capable of immersing atleast one section of the sample in the liquid accumulated in thesubstrate.

With this configuration, the sample and the flat surface section of thesubstrate are brought into contact with each other in a state in whichat least one section of the sample is immersed in the liquid accumulatedin the substrate, thus making it possible to suppress drying of thesample during observation.

In the above-described third aspect, the contact-state maintaining unitmay be a positioning mechanism capable of positioning the base materialin a state in which the sample and the flat surface section are broughtinto contact with each other.

With this configuration, due to the positioning mechanism, it ispossible to ensure a sufficient contact area between the sample and thesubstrate while the base material maintains the sample in a stableorientation.

In the above-described third aspect, the positioning mechanism may beconfigured so as to be capable of positioning the sample and the flatsurface section at at least two relative positions in a contact stateand a non-contact state.

With this configuration, due to the positioning mechanism, it ispossible to bring the sample and the flat surface section of thesubstrate into contact with each other and to maintain the state, withease and accuracy.

In the above-described third aspect, a plurality of substrates may bearranged in an array.

With this configuration, it is possible to observe and acquire images ofa plurality of observation targets at once, thus making it possible toachieve the efficiency of observation and image acquisition.

According to the present invention, an advantageous effect is affordedin that it is possible to get the best from the original opticalperformance of the microscope, to enable high-definition observation andimage acquisition of a cellular aggregate performed by the microscope,and to facilitate automation of the observation.

REFERENCE SIGNS LIST

-   1 hanging-drop forming device (base material)-   3, 3″ well (substrate)-   3 a bottom section (flat surface section)-   5 positioning mechanism (contact-state maintaining unit)-   7, 33 sample holder-   9 objective lens-   23 notch mechanism (positioning mechanism, contact-state maintaining    unit)-   25 slide glass (substrate)-   25 a flat surface section-   31 pressing member (contact-state maintaining unit)-   B droplet (sample)-   D hanging drop (sample)-   S cellular aggregate (observation target)-   W1 transparentizing solution (liquid)

The invention claimed is:
 1. A sample observation method comprising:bringing a gel-like transparent sample that encloses an observationtarget into contact with a transparent flat surface section of asubstrate; and collecting light from the observation target by means ofan objective lens of a microscope via the substrate, in a state in whicha pressing force is made to act in a direction in which the sample andthe flat surface section relatively approach each other, until a contactarea between the sample and the flat surface section comes to have asize allowing an effective light flux for the objective lens to passtherethrough, wherein the pressing force is made to occur due togravity.
 2. The sample observation method according to claim 1, whereina base material supports the sample in a state in which the sample isbrought into contact with the flat surface section.
 3. The sampleobservation method according to claim 1, wherein the microscope is aninverted microscope.
 4. The sample observation method according to claim1, wherein the objective lens is an immersion objective lens.
 5. Thesample observation method according to claim 1, wherein the sample isformed by causing a transparent droplet to gel in a state in which theobservation target is enclosed therein.
 6. The sample observation methodaccording to claim 1, wherein the observation target is a cellularaggregate formed when a plurality of cells aggregate.
 7. A sampleobservation method comprising: bringing a gel-like transparent samplethat encloses an observation target into contact with an objective lensof a microscope; and collecting light from the observation target bymeans of the objective lens, in a state in which a pressing force ismade to act in a direction in which the sample and the objective lensrelatively approach each other, until a contact area between the sampleand the objective lens comes to have a size allowing an effective lightflux for the objective lens to pass therethrough, wherein the pressingforce is made to occur due to gravity.
 8. The sample observation methodaccording to claim 7, wherein the microscope is an inverted microscope.9. The sample observation method according to claim 7, wherein theobjective lens is an immersion objective lens.
 10. The sampleobservation method according to claim 7, wherein the sample is formed bycausing a transparent droplet to gel in a state in which the observationtarget is enclosed therein.
 11. The sample observation method accordingto claim 7, wherein the observation target is a cellular aggregateformed when a plurality of cells aggregate.
 12. A sample observationmethod comprising: bringing a gel-like transparent sample that enclosesan observation target into contact with a transparent flat surfacesection of a substrate; and collecting light from the observation targetby means of an objective lens of a microscope via the substrate, in astate in which a pressing force is made to act in a direction in whichthe sample and the flat surface section relatively approach each other,until a contact area between the sample and the flat surface sectioncomes to have a size allowing an effective light flux for the objectivelens to pass therethrough, wherein the sample is dropped on thesubstrate, thus being brought into contact with the flat surfacesection.
 13. The sample observation method according to claim 12,wherein a base material is vibrated from a state in which the basematerial supports the sample, thereby dropping the sample.
 14. A sampleobservation method comprising: bringing a gel-like transparent samplethat encloses an observation target into contact with a transparent flatsurface section of a substrate; and collecting light from theobservation target by means of an objective lens of a microscope via thesubstrate, in a state in which a pressing force is made to act in adirection in which the sample and the flat surface section relativelyapproach each other, until a contact area between the sample and theflat surface section comes to have a size allowing an effective lightflux for the objective lens to pass therethrough, wherein the substratehas a form capable of accumulating a liquid therein; and wherein thesample and the flat surface section are brought into contact with eachother in a state in which at least one section of the sample is immersedin liquid accumulated in the substrate.
 15. The sample observationmethod according to claim 14, wherein the liquid accumulated in thesubstrate is a transparentizing solution for making the sampletransparent.
 16. A sample observation method comprising: bringing agel-like transparent sample that encloses an observation target intocontact with a transparent flat surface section of a substrate; andcollecting light from the observation target by means of an objectivelens of a microscope via the substrate, in a state in which a pressingforce is made to act in a direction in which the sample and the flatsurface section relatively approach each other, until a contact areabetween the sample and the flat surface section comes to have a sizeallowing an effective light flux for the objective lens to passtherethrough, wherein the sample is formed by causing a hanging drop togel, the hanging drop being a transparent droplet, in which theobservation target is enclosed, in a state in which the droplet is hung.17. A sample observation method comprising: bringing a gel-liketransparent sample that encloses an observation target into contact withan objective lens of a microscope; and collecting light from theobservation target by means of the objective lens, in a state in which apressing force is made to act in a direction in which the sample and theobjective lens relatively approach each other, until a contact areabetween the sample and the objective lens comes to have a size allowingan effective light flux for the objective lens to pass therethrough,wherein the sample is formed by causing a hanging drop to gel, thehanging drop being a transparent droplet, in which the observationtarget is enclosed, in a state in which the droplet is hung.
 18. Asample holder comprising: a substrate that has a transparent flatsurface section with which a gel-like transparent sample that enclosesat least one observation target is brought into contact; a contact-statemaintaining unit that maintains a contact state in which a contact areabetween the sample and the flat surface section has a size allowing aneffective light flux for an objective lens of a microscope to passtherethrough; and a base material that supports the sample in a state inwhich the sample is brought into contact with the flat surface section,wherein the base material can support a droplet in a hung state, withthe droplet enclosing the observation target; and the sample is formedby causing the droplet, which is supported by the base material, to gel.19. The sample holder according to claim 18, wherein the contact-statemaintaining unit comprises a pressing member that presses the sample ina direction in which the sample is pressed against the flat surfacesection.
 20. The sample holder according to claim 18, wherein thecontact-state maintaining unit comprises a positioning mechanism capableof positioning the base material in a state in which the sample and theflat surface section are brought into contact with each other.
 21. Thesample holder according to claim 20, wherein the positioning mechanismis configured so as to be capable of positioning the sample and the flatsurface section at at least two relative positions in a contact stateand a non-contact state.
 22. The sample holder according to claim 18,wherein a plurality of substrates are arranged in an array.
 23. A sampleholder comprising: a substrate that has a transparent flat surfacesection with which a gel-like transparent sample that encloses at leastone observation target is brought into contact; a contact-statemaintaining unit that maintains a contact state in which a contact areabetween the sample and the flat surface section has a size allowing aneffective light flux for an objective lens of a microscope to passtherethrough; and a base material that supports the sample in a state inwhich the sample is brought into contact with the flat surface section,wherein the substrate has a form capable of accumulating a liquidtherein and capable of immersing at least one section of the sample inliquid accumulated in the substrate.